LILAR, a novel long noncoding RNA regulating autophagy in the liver tissues of endotoxemic mice through a competing endogenous RNA mechanism

Abstract Sepsis is an often‐deadly complication of infection that can lead to multiple organ failure. Previous studies have demonstrated that autophagy has a protective effect on liver injury in sepsis. Here, we report a novel long noncoding RNA (lncRNA), named lipopolysaccharide (LPS)‐induced liver autophagy regulator (LILAR), which was highly induced in the liver tissues of endotoxemic mice. LILAR deficiency significantly increased the susceptibility of mice to LPS. In contrast, LILAR overexpression rescued the liver injury mediated by LILAR deficiency and increased the survival of LILAR knockout mice with endotoxemia. Autophagy‐related protein 13 (Atg13) is a potential downstream target gene of LILAR. LILAR deficiency notably decreased Atg13 expression and suppressed autophagy in the livers of mice challenged with LPS. A reporter gene assay showed that LILAR competitively adsorbed miR‐705 to increase the expression of Atg13 in cultured cells, indicating that LILAR participates in the regulation of the autophagy in the liver tissues of endotoxemic mice through a competitive endogenous RNA mechanism. In summary, we identified a novel lncRNA, LILAR, as a hepatic autophagy regulator, which not only promotes our understanding of liver pathophysiology but also provides a potential therapeutic target and/or diagnostic biomarker for liver injury in endotoxemia.


RNA-seq
A total RNA isolation kit (Biomarker, #RK02009) was used to isolate RNAs from liver tissues following the illustrations of the manufacturer.Before further experiments, the integrity, purity, and concentration of RNA were monitored and assessed to meet the subsequent requirements.For rRNA removal, the Ribo-Zero rRNA removal kit from Epicenter (Madison, WI, USA) was used.
The Next Ultra Directional RNA Library Prep Kit (New England Biolabs) was utilized to generate the sequencing libraries following the illustrations of the manufacturer.After cluster generation by the TruSeq PE Cluster Kit (v3-cBot-HS), an Illumina HiSeq 4000 platform was used to sequence the library preparations.

Cell migration assay
We performed cell migration assays according to previous reports. 2Corning Costar transwell plates (Corning, #3422, New York, USA) were used for cell migration assays according to the manufacturer's protocol.BMDMs were seeded in the upper chambers with FBS-free DMEM media.The lower chamber was filled with DMEM containing 10% FBS as a chemo attractant.The cells were cultured at 37 °C in 5% CO 2 for 48 h.Then, the upper chambers were removed and the non-migrated BMDMs were wiped away.The 4% paraformaldehyde was used to fix migrated cells.After being stained with crystal violet (Beyotime, #C0121, Shanghai, China), the cells were observed under a microscope of EVO XL core AMEX1000 imaging system (ThermoFisher, Wilmington, DE, USA) and images were taken for cell counting with the cell counter plugin of the ImageJ software (Version 1.53u).

Phagocytosis assays
Phagocytosis assays were performed by pHrodo™ Red Zymosan Bioparticles (ThermoFisher, #P35364) according to the manufacturer's instructions.Briefly, Zymosan BioParticles conjugates of 0.5 mg/mL were resuspended for the medium replacement of the phagocytic cells.After incubation for 30 min, cells were acquired for analysis with the software (IDEAS, Version 6.0) on an ImageStream system (EMD Millipore, Washington, USA).

Protein extraction from liver tissues and cultured cells
RIPA buffer (ThermoFisher Scientific, #89900) with protease inhibitors (Roch, #11697498001) was utilized to extract proteins from liver tissues or HEK293T cells.Briefly, the tissue samples frozen in liquid nitrogen (approximately 5 mg) were crushed in a mortar and pestle.The samples were resuspended by RIPA buffer and sonicated with a VCX800 ultrasonic cell homogenizer (Sonics, CT, USA).HEK293T cells were collected by centrifugation at 500 × g for 5 min at 4°C, followed by resuspension with 300 µL of RIPA buffer and sonication as above.After centrifugation at 12,000 × g for 25 min at 4°C, the supernatant was collected for quantification of protein concentration by the bicinchoninic acid (BCA) method.

Histopathological examination
Liver tissues were isolated from the left lobe and the blood was removed by washing thrice in PBS.Following fixation in 4% paraformaldehyde (Biosharp, #BL539A) and subsequent paraffin embedding, slices of liver tissues were prepared for H&E staining.The injury score in each field was quantitated according to previous reports. 3

Cytokine/chemokine quantitation
Quantification of serum cytokines and chemokines was performed using the LiquiChip system from Qiagen (Hilden, Germany) according to our previous reports. 4

Isolation of total RNA and qRT-PCR
The manufacturer's protocol was followed for the isolation of total RNA from cells or tissues using TRIzol reagent (#15596026) supplied by Invitrogen (Carlsbad, CA, USA).The RNA concentration and quality were assessed using a NanoDrop spectrophotometer from ThermoFisher Scientific, USA.The reverse transcription of mRNA was performed by a ReverTra Ace TM qPCR RT Kit (#FSQ-101, TOYOBO, OSA, Japan) following the manufacturer's protocol.For microRNA, miR-705 was reversely transcribed using specific primers from RiboBio Co., Ltd.(Guangzhou, China).The Applied Biosystems TM 7500 real-time PCR system (CA, USA) was used to perform qRT-PCR analysis.The primers for mRNA are listed in Table S1.The Bulge-Loop miRNA qRT-PCR Primer Sets (RiboBio) were used for miR-705 (#MQPS0003068-1) and U6 (#MQPS0000002-1-100).The 18S rRNA and U6 were used as internal controls for the quantitation of mRNA and miRNA, respectively.The relative expression level of a gene was assessed utilizing the 2 -ΔΔCT comparative method.

Flow cytometry analysis
Flow cytometry was performed as previously reported. 5Splenic single cells were incubated with specific antibodies (Table S3) for staining at 4°C.After washing with PBS, cells were acquired for analysis with EDAS software (Version 6.0) on an ImageStream system (EMD Millipore, Washington, USA).

Transmission electron microscopy
Liver tissues were fixed with 2.5% glutaraldehyde at pH 7.4 for 1 h and processed for transmission electron microscopy (TEM) detection as previously described. 6Briefly, after fixation and dehydration, an MT-6000-XL-RMC ultramicrotome from Boeckeler Instruments, Inc (Tucson, AZ) was used to obtain ultrathin sections (70-80 nm).Then, the sections were stained with uranyl acetate and lead citrate and examined with an H-7500 transmission electron microscope from Hitachi, Ltd (Tokyo, Japan).

Immunohistochemistry
Immunohistochemical analysis was performed according to previous reports. 7Briefly, after deparaffinization and rehydration, the citrate buffer was used for antigen retrieval of the mouse liver tissue slices.Phosphate-buffered saline (PBS) containing 3% H 2 O 2 was used to block endogenous peroxide.After blocking with 10% bovine serum albumin (BSA), the sections were incubated with monoclonal LC3B antibody (NOVUS, #NB100-2220) at 4°C overnight.After being treated with an avidin-biotin affinity system, the slices were stained with 3,3′-diaminobenzidine (DAB) and hematoxylin for microscopic examination.The obtained pictures were analyzed by Image-Pro Plus (Version 6.0.0260).

Western blotting
Western blotting was performed as reported previously. 8Samples containing equal amounts of proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) membranes from Millipore (#IPVH00010).The membranes were blocked with 5% BSA buffer (#V900933) from Sigma Aldrich (Darmstadt, Germany) and then incubated with the specific primary antibodies mentioned above.After 3 washes for 5 min with a Washing Buffer, i.e.Tris-buffered saline containing 0.1% Tween-20 (TBST), the membrane was incubated with horseradish peroxidase (HRP)-conjugated anti-mouse or rabbit secondary antibody.The protein bands were visualized by enhanced chemiluminescence (ECL) (ThermoFisher Scientific, #32109), and the images were captured using a ChemiDoc™ Imaging System provided by Bio-Rad (Hercules, CA, USA).
The transfection concentration was 300 ng for the pGL3 luciferase reporter vector, 10 ng for the pRL-CMV vector, and 50 nM for miRNA mimics.A Dual-Luciferase Reporter Assay System (Promega, Madison, Wis, USA ) was utilized to perform luciferase assays.Ratios of Firefly luciferase and Renilla luciferase readings were evaluated in triplicate in each group.

RNA pulldown
RNA pulldown experiments were performed by using biotinylated LILAR (Bio-LILAR), Atg13-3'UTR (Bio-Atg13-3'UTR), and negative control (Bio-NC) based on previously described methods with some minor modifications.The biotinylated probes were prepared using a T7 RNA polymerase kit (Roche, #10881767001) following the manufacturer's instructions.RNA extracted from mouse livers was incubated overnight with a biotinylated probe of LILAR, Atg13-3'UTR, or negative control at 4°C.Subsequently, streptavidin-coated magnetic beads (ThermoFisher Scientific, #PI88817) were added and incubated for 2 h at 4°C.After 3 to 5 washes of the beads with incubation buffer, miR-705 in the pulldown mixture was measured by qRT-PCR.

Figure S4 .Figure S5 .Figure S6 .Figure S7 .Figure S8 .Figure S9 .Figure S10 .
Figure S1.Assessment of liver injury model of mice induced by LPS.Mice were intraperitoneally injected with LPS (20 mg/kg) for 12 h or an equal volume of normal saline (NS) for control.(A) Protein quantitation of IL-6, IL-1β, and TNF in the serum of mice treated without or with LPS for 12 h.n = 3; *P < 0.05, compared with the control group.(B) The levels of ALT, AST, and LDH in the serum of mice treated without or with LPS for 12 h.n = 3; *P < 0.05, compared with the control group.(C) Histopathological evaluation of liver injury.H&E staining was performed as described in the methods and the amount of infiltration of inflammatory cells, hemorrhage, and hepatocyte necrosis were used as parameters to assess the liver injury.n = 4; *P < 0.05, compared with the control group.(D) The 24-hour survival rate of mice challenged with LPS.Statistical analyses were performed using the log-rank (Mantel-Cox) test.n = 10 for each group; *P <0.05, compared with control group.